Intravital imaging of CTLs killing islet cells in diabetic mice Ken Coppieters, Natalie Amirian, Matthias von Herrath
Published in Volume 122, Issue 1 J Clin Invest. 2012; 122(1):119–131
1. “Random walk” in acinar pancreas of CD8 T cells targeting induced beta cell viral protein (LCMV-GP).
2. CD8 T cells arrest at post-capillary venules. 3. Killing may require 6 hour CD8 beta cell contact.
Prevention of Diabetes in TCR Transgenic anti-IGRP206-214 (CD8) NOD Mice by tolerizing to proinsulin.
Krishnamurthy et al J. Immunol 2008, 180:4458-4464.
0
0.2
0.4
0.6
0.8
1
1.2
'0 '3 6+
Beta Cell Area
Weeks after anti-CD3 mAb Therapy NOD MiceSherry et al, Effects of Autoimmunity and Immune Rx on B-Cell Turnover in Type 1 Diabetes. Diabetes 55:3238-3245
% Ki67+ beta cells 4.8% 2.5% 1.2%
Initial non-DM non-DM non-DMpost neph
Boston glucose>400Nishio et al
2006 Tx+CFA+Spleen 30 9 5 No islet splencoyte derived cellsTx+CFA 13 4
Chicago glucose >300 for 2 daysChong et al
2006 Tx+CFA+Spleen 22 7 6 No islet splenocyte derived cellsperi-insulitis
St. Louis Hyperglycemia; Insulin Rx 7-20 daysSuri et al Tx+CFA+Spleen 53 22 4 No Y-chromosome, No chimerism by flow
2006 Tx+CFA 29 20 No GFP+ islet,
Mass Gen Tx+CFA+Spleen 21 20 17 Y-choromsome+, GFP+ Kodma et al
2003
Diabetes Studies Conflict on Power of Spleen Cells: Jennifer Couzin, Science 24 March 2006, Vol 311: 1694
Turvey et al: Noninvasive imaging of pancreatic inflammation and its reversal in
type 1 diabetes JCI 115:2454, 2005
202224262830323436
New Onset Non-DM NOD E alpha NOD
T2(ms)
Mordes et al: LEW.1WR1 Rats Develop Autoimmune Diabetes Spontaneously and in
Response to Environmental Perturbation Diabetes 54:2727, 2005
0102030405060708090
100
No Rx Poly-IC Anti-ART2.1
% Diabetic
Rats are MHC Congenic Lewis with RT1 AuB/Du/Ca thus “diabetogenic” class II, and small % insulitis diabetes w/o poly-IC.
Devendra et al: Interferon-alpha as a Mediator of Polyinosinic:Polycytidylic Acid Induce Type 1
Diabetes Diabetes 54:2549, 2005
r = - 0.58,p<0.01
0
50
100
150
200
250
300
350
8 13 18 23 28 33 38
Age of diabetes onset (weeks)
IFN alpha (pg/ml)
Age of diabetes Onset (weeks)
Serum Interferon post poly-IC (pg/ml)
Poly-IC induction diabetes in RIP-B7.1 mouse model acts through interferon alpha, with antibody blocking, levels correlating (above) and interferon itself inducting DM.
Spontaneous Animal Models BB rat
Homozygosity Lymphopenia (Ch4), Ian4 gene mutation
RT1-U class II (Ch 20)Additional Loci (Ch2,18,X)
NOD mousePolygenic: class II + class I loci + IL-2 linked
polymorphism + >12 Long-EvansTokushima Rat (Komeda Diabetes Prone)
RT1-U MHCHomozygosity Chromosome 11, Cblb mutation
LEW.1AR1/Ztm-iddm ratRT1-U MHC for class II B/D, Cu but Aa
Human DQ8 with islet B7-1 Transgene (RIP-B7-1)B7-1 costimulator (Wen et al.)
BDC-Jun02
NOD Mice
Develop Type 1A-Immune Mediated Diabetes
Are inbred and thus identical at all genetic loci
Genetic loci from other mice can be backcrossed by sequential breeding to fix genes that might influence development of diabetes
“Families” of Hundreds of Identical Twins
Insulitis at 5 weeks diabetes at 16-30 weeks
Spontaneously develop autoimmune diabetes
Nonobese Diabetic (NOD) Mice
Females afflicted more commonly than males
outbredICR mice
NOD
NON
cataractsCTS
(cataractShionogi)
F6
F20
diabeticnormal fastingblood glucose
high fastingblood glucose
X
Origin:
T. DiLorenzo
Other NOD Characteristics
Defects in differentiation and function of APCs
Deficiency in CD4+CD25+ regulatory T cells
NK T cell deficiencies (number and function)
Defective NK cell activity
Lack serum hemolytic complement activity (no C5)
I-Enull
Impaired production of IL-4
Defects in FcgRI and FcgRII
b2-microglobulin and CTLA-4 are susceptibility genes
T. DiLorenzo
Other Genes Insulin Gene VNTR Type 1A Diabetes
Protection with greater thymic messenger RNA
AIRE gene APS-I syndromeAutosomal recessive: 18% Diabetes
Scurfy gene of XPID SyndromeNeonatal death overwhelming autoimmunity
Ian 4/5 recessive lymphopenia gene BB rat
Cblb recessive autoimmune gene LETL rat
Multiple loci unkown significance
Rat Strains with Spontaneous or Induced type 1 Diabetes
I RT1 II
I
Rat Strain A B/D C Diabetes BB-DP U U U Spontaneous LETL U U U Spontaneous BB-DR U U U KRV DM Lew1.WR1 U U A Poly-IC DM Lew1.AR1 A U U Spontaneous PVG.RT1u U U U Poly-IC DM PVG.R8 A U U Poly-IC DM WF U U U 1/15 Poly-IC DM WAG U U U 1/9 Poly-IC DM
Ellerman et al. Diabetologia 2,000; Whalen et al. Transplant Proc: 199729:1684-5;Lenzen et al. Diabetologia 2001BDC
The BB Diabetic Rat: Profound T-Cell LymphopeniaJackson, Rassi, Crump, Haynes and Eisenbarth
Diabetes 30: 887-889, 1981
0123456789
10
% B lymph % Non-Blymph
#W3/25 /1000T Cells
BB ratWistar
BDC
Two Genes Required for Diabetes in BB Rats
0
10
20
30
40
50
60
% W
3/25
T C
ells
Diabetic
Non-Diabetic
Intercross Lewis BN Wistar //BackcrossJackson et al J. Exp Med, 159:1629-1636, 1984 BDC
Immune-Associated Nucleotide-Related: Ian-4(5) gene: BB rat
lymphopenia Rat Chromosome 4, within 290Kb region of
lymphopenia locus BB rat GTP binding protein outer mitochrondrial membrane Hypothesized to protect from apoptosis Expressed spleen and thymus Frameshift mutation BB (450delC) Ian-4bb last 215 amino acids missing, replaced by 19
other amino acids, including lost membrane binding region
Autosomal recessive determinant severe lymphopenia of BB rat necessary for spontaneous diabetes
Markholst et al, Diabetes 51:1972-1979, 2002MacMurray et al, Genome Res 2002, 12:1029
Cblb: (Casitas B-lineage lymphoma b) Autosomal Recessive Diabetogene of Komeda/LETL Rat Cblb Mice development generalized autoimmunity LETL/Komeda Rat nonsense mutation, stop codon
removing 484 amino acids including leucine zipper and proline rich region
Transgenic Replacement Cblb Prevents Diabetes Homologous human gene on Chromosome 3 T cells Cblb deficient mice do not require CD28 for
activation and Vav1 highly activated independent of CD28 costimulation
Yoikoi et al. Nature Genetics 31:391-394, 2002
The non-obese diabetic (NOD) mouse
An inbred strain of mice with spontaneous development of autoimmune type 1 diabetes
The cumulative incidence of diabetes: 80% in females, 50% in males (at 30 weeks of age)
Both MHC and non-MHC genes are required for development of the disease
H. Ikegami
The NOD mouse: recessive diabetogenic gene within the major histocompatibility complex
Hattori et al. Science 231:733-735, 1986
BDC
Idd1
Idd3Idd10Idd18.2
Idd5.1
Idd5.2
Idd9.1
Idd9.3HLA
IDDM12
IDDM2
IDDM15
IDDM5
IDDM8
IDDM10
IDDM17
IDDM416q24
16pXP11
Genes in Human & NOD Type 1 Diabetes/2004Provided by J Todd & L Wicker
For more information visit http://www.t1dbase.org/cgi-bin/welcome.cgi
HLA CLASS II& others?
INSULIN
CTLA-4(both species)
4-1BB
CD101
VAV3 IL-2
PTPN22 in humans, Ptpn8 in NOD
NOD MHCCLASS II &other loci
NRAMP1
Idd18.1
Idd9.2
IL2RA
Low incidence of type 1 diabetes in NOD mice congenic for Idd3 region of
chromosome 3 from B6 strain
Chr2
34
56
78
910
112
1314
151617
1819
X
11
20% Wicker LS et al. J Exp Med 1994Lyons PA et al. Genome Res 2000
B6
.B6-Idd3
NOD
1%
B6
NOD.B6-chr3
80%
The NOD mouse and its related strains
Jcl:ICR CTSNOD
NON
(outbred)
NCT
NSY
IISILIIOI
H. Ikegami
NOR
B-cell Mass (mg) NOD vs NOD SCIDSreenan et al; Diabetes 48:989
00.20.40.60.8
11.21.41.6
8-9 wks 13 wks 18 wks
%DM 0 11% 70%
NOD SCID
NOD
BDC
Identification of Insulin but Not Glutamic Acid
Decarboxylase or IA-2 as Specific Autoantigens of
Humoral Autoimmunity in Nonobese Diabetic Mice
Bonifacio et al Diabetes 50:2451-2458, 2001
International Workshop on Lessons From Animal Models for Human Type 1
Diabetes
4 8 12 16 20 24 28 32 36
0.001
0.01
0.1
1
10
0
200
400
600
800
weeks
IAA (index) BS (mg/dl)
4 8 12 16 20 24 28 32 36
0.001
0.01
0.1
1
10
0
200
400
600
800
weeks
IAA (index) BS (mg/dl)
4 8 12 16 20 24 28 32 36
0.001
0.01
0.1
1
10
0
200
400
600
800
weeks
IAA (index) BS (mg/dl)
4 8 12 16 20 24 28 32 36
0.001
0.01
0.1
1
10
0
200
400
600
800
weeks
IAA (index) BS (mg/dl)
4 8 12 16 20 24 28 32 36
0.001
0.01
0.1
1
10
0
200
400
600
800
weeks
IAA (index) BS (mg/dl)
4 8 12 16 20 24 28 32 36
0.001
0.01
0.1
1
10
0
200
400
600
800
weeks
IAA (index) BS (mg/dl)
4 8 12 16 20 24 28 32 36
0.001
0.01
0.1
1
10
0
200
400
600
800
weeks
IAA (index) BS (mg/dl)
4 8 12 16 20 24 28 32 36
0.001
0.01
0.1
1
10
0
200
400
600
800
weeks
IAA (index) BS (mg/dl)
4 8 12 16 20 24 28 32 36
0.001
0.01
0.1
1
10
0
200
400
600
800
weeks
IAA (index) BS (mg/dl)
4 8 12 16 20 24 28 32 36
0.001
0.01
0.1
1
10
0
200
400
600
800
weeks
IAA (index) BS (mg/dl)
4 8 12 16 20 24 28 32 36
0.001
0.01
0.1
1
10
0
200
400
600
800
weeks
IAA (index) BS (mg/dl) 4 8 12 16 20 24 28 32 36
0.001
0.01
0.1
1
10
0
200
400
600
800
weeks
IAA (index) BS (mg/dl)
4 8 12 16 20 24 28 32 36
0.001
0.01
0.1
1
10
0
200
400
600
800
weeks
IAA (index) BS (mg/dl)4 8 12 16 20 24 28 32 36
0.001
0.01
0.1
1
10
0
200
400
600
800
weeks
IAA (index) BS (mg/dl)
4 8 12 16 20 24 28 32 36
0.001
0.01
0.1
1
10
0
200
400
600
800
weeks
IAA (index) BS (mg/dl)
WEEKS
IAA
leve
lsB
lood Suga r leve ls
GLUCOSEINSULIN AbBY AGE NOD
BDC
Inhibition of NOD Diabetes in Absence of Transplacental Antibodies (Ab)Greeley et al, Nature Med 8:399, 2002
01020304050607080
IgMKnockout
Anti-HEL+KO
DBA/2FosterMother
SCIDMother
Control No Maternal Ab
Autoantibodies/Autoreactive B Cells Contribute to NOD Diabetes
Immunoglobulin knockout prevention NOD DMSerreze et al, J. Immunol 1998, 161:3912-3918
I-Ag7 on B cells needed for NOD diabetes.Noorchashm et al, J. Immunol 1999, 163, 743-750
Anit-Insulin VH125 Heavy Chain Increases diabetes in NOD mice.Hulbert et al, J. Immunol, 2001, 167: 5535-5538
Transplacental autoantibodies accelerate NOD diabetes.Greeley et al, Nature Medicine, 8:399, 2002
B Cell Deficient Child Developed Type 1A DiabetesMartin et al, NEJM, 2001, 345:1036-1040
BDC
Reactivity of B:9-23 reactive T cell clones to truncated peptides
BDC12-2.40 BDC 14-4.1BDC 12-4.4BDC 6-4.3BDC 6-11.6
+ ++ -+ -+ -- -- -- -- -- -- +- +
B:9-23 S H L V E A L Y L V C G E R G
B:9-23 (15)
B:9-20
B:9-17
B:9-16 (8)
B:9-15
B:9-14
B:10-19
B:15-23
B:14-23
B:13-23 (11)
B:12-23
BDC
Unique properties of the insulin B chain peptidein NOD islet derived CD4 and CD8 T cell clones
1) Insulin Peptide B:9-23Majority islet CD4 cells recognizeT cells transfer diseasePrevents disease
2) AV13S3, AJ53 or AJ42 Restriction3) Dual Overlapping Peptides (B:9-16 and B:13-23) Recognized by
AV13S3AJ52TCR T Cell Clones
4) Insulin Peptide B:15-23 Recognized by pathogenic CD8 T cell clone from NOD mice
A high percentage of Kd CD8 T cells recognize
1) D. Wegmann et al. (1994) Eur J Immunol 24,1853-1857 etc.2) Eric Simone et al. (1997) Proc Natl Acad Sci USA 94,2518-25213) Abiru N. et al.(2000) J Autoimmune 14:231-2374) F. Susan Wong et al. (1999) Nature Medicine5.9:1026-1031
BDC
Subcutaneous Injection of B:9-23
0
20
40
60
80
100
0 10 20 30 40 50Age (weeks)
Perc
ent n
on-
Diabe
tic
B:9-23 Peptide
BDC
InductionInsulin
Autoantibodies/Insulitis/Diabetes
B:9-23 Peptide ----- Insulin Autoantibodies B:9-23 Peptide + Poly-IC ------ Insulitis
B:9-23 Peptide + Poly-IC + B7.1 Islet -- Diabetes
Moriyama et al. PNAS 99: 5539-5544, 2002
Experimental Autoimmune Diabetes:H-2d (of Balb/c)+Insulin B:9-23
H-2d B:9-23 Poly-IC Islet B-7.1
IAA Insulitis Diabetes
+ + - - Yes No No
+ + + - Yes Yes No
+ + + + Yes Yes Yes
+ + - + Yes Yes Yes
+ - + + Low Yes Yes
- + + - No No No
Moriyama et al, PNAS 99: 5539-5544, 2002
BDC
Rapid induction of IAA by Insulin B:9-23 peptide Imunization in Normal BALB/c mice
3 4 5 6 7 8 9 10 11 12 13
0.001
0.01
0.1
1
10
weeks
IAA (index)
B:9-23+ IFA B:9-23+ IFABDCAbiru et al Diabetes 50:1274-1281, 2001
a b
c d
PNAS 99:5539-5544
Blood glucose level in B7-1, H-2d miceTT in IFA or IFA + Poly-IC (DM, 12/16)B:9-23 in IFA + Poly-IC (DM, 9/9)
0
100
200
300
400
500
600
0 4 8 12 16 20 24 28 32(Weeks of age)
0
100
200
300
400
500
600
0 4 8 12 16 20 24 28 32
(mg/ml)
(Weeks of age)
(mg/ml)
B:9-23 in IFAPoly-IC
TT in IFA or IFA alonePoly-IC
PNAS 99:5539-5544
Immunohistochemical Staining in H-2d mice:Immunized with B:9-23+poly-IC
CD4 CD8
B7-
B7+
PNAS 99:5539-5544
CYTOKINE DEPENDENCY OF NON-Th2 REGULATORY T CELLS
CD45RBhi T-cell induced colitisday 3 ThymectomyThymectomy-Radiation (rat)NODNKT cells
-
-
+
-
+
+
-
?
-
+
+
?
+
?
?
IL-4 IL-10 TGFbExperimental model
Bach
Insulin Peptide Induction Anaphylaxis Liu et al. JCI 2002
Insulin B:9-23 in saline – 7 injections = death NOD
Anaphylaxis dependent upon bothIgG and IgE antibodiesHistamine and Platelet Activating Factor
Anaphylaxis following subcutaneous injection prevented with addition RR to peptide to produce peptide with neutral pI while peptide able to prevent diabetes of NOD mice
Peri-Islet Schwann Cells (pSC) and NOD MiceDosch et al Nature Med 2003;9:198-205
Express GFAP and S100 beta
Destroyed NOD mice, TCR transgenic 8.3 (anti-NRP) but not LCMV TCR model
Autoantibodies with mass spec assay
T Cell responses (low level)
T cell clones to GFAP, perinsulitis but no diabetes
Acceleration of type 1 diabetes mellitus in proinsulin 2-deficient
miceThebault-Baumont et al JCI 111:851, 2003
Preproinsulin 2 gene knockout bred onto NOD mouse accelerates diabetes
-/- mice have greater insulin autoantibodies(no difference GAD Ab but ?Ab ELISA artifact given workshop data)
Increased insulitis -/- female mice at 8 weeks of age
Preproinsulin 2/1 peptide 88-103 recognized post immunization insulin 2-/- but not +/+ mice (KRGIVDQCCTSICSLY [in A chain])
Normal Incidence of Diabetes in NOD Mice Tolerant to
Glutamic Acid Decarboxylase
E. Jaeckel et al.J Exp. Med 197:1635-1644, 2003
“Our experiments suggest that the protection observed in the GAD-antisense experiments
has no immunologic basis.”
insulin 1 KO male
0 10 20 30 40 500
20
40
60
80
100
weeks of age
% o
f dia
bete
s fr
ee
Insulin 1 KO female
0 10 20 30 40 500
20
40
60
80
100
weeks of age
% o
f dia
bete
s fr
ee Insulin 2 KO male
0 10 20 30 40 500
20
40
60
80
100
weeks of age
% o
f dia
bete
s fr
ee
Insulin 2 KO female
0 10 20 30 40 500
20
40
60
80
100
weeks of age
% o
f dia
bete
s fr
ee
PNAS 2003,18:10376PNAS: 2003, 18:10376
Hematopoetic Stem Cells-Proinsulin(PI) Prevent NOD Diabetes
0
10
20
30
40
50
60
0 50 100 150 200 250 300
Age (days): Rx 4 weeks
Perc
ent D
iabe
tic HSC-PI
HSC
No Rx
Steptoe et al, JCI 2003:111:1357
Creation of Surviving NOD Mice Lacking Native Insulin Sequence B:9-23
See Makayama et al. Prime role for an insulin epitope in the development of type 1 diabetes in NOD mice Nature 435:220, 2005
0 10 20 30 40 50 600
20
40
60
80
100
ins1-, ins2-, Tg+ (n=25, P<0.01)ins1+, ins2-, Tg+ (n=25)
Weeks of age
% D
iabe
tes
Free
Lack of progression to diabetes of NOD mice lacking both insulin native genes.
Ins1-, ins2-: n=Ins1+, ins2-: n=
2525
1014
2123
11
24 Life table update 5/19/05
Normal Histology of native insulin-negative NOD mouse with B16:alanine mutated insulin transgene
Insulin Staining
See Makayama et al. Prime role for an insulin epitope in the development of type 1 diabetes in NOD mice Nature 435:220, 2005
Splenocytes from native insulin-negative mice can induce diabetes into NOD.SCID mice but with delay potentially related to recapitulation attack
on islets with native insulin B:9-23 sequence.
ins1-/-, ins2-/-, tg+
splenocytes
NOD-SCIDIns1+/+, ins2+/+
Diabetes!!No diabetes
0 10 20 300
20
40
60
80
100
ins1+, ins2+, Tg- (n=8)ins1-, ins2-, Tg+ (n=13, P<0.01)
Weeks post transfer
% D
iabe
tes
Free
Life table update 5/19/05
Transfer from NOD-PI mice of hematopoietic stem cells encoding proinsulin expression by MHC class II+ progeny prevents diabetes
Steptoe RJ, Ritchie JM, Harrison LC (2003) Transfer of hematopoietic stem cells encoding autoantigen prevents autoimmune diabetes. J Clin Invest 111:1357-1363.
0 100 200 3000
10
20
30
40
50
60
70
NOD wild type HSCrecipients
NOD proinsulin HSCrecipients
NOD colony
Inci
denc
e of
dia
bete
s (%
)
Age (days)
1x103 HSC (lin-, SCA-1+, c-kit+) i.p. to irradiated recipients at 4 weeks of age
Recipients ofwild-type NOD HSCs
Recipients ofNOD-PI HSCs
Harrison